Pharmaceutical composition active in reducing production of MCP-1 protein

ABSTRACT

The present application relates to a method of treating atherosclerosis by administering to a patient a compound of formula:                    
     and/or salts thereof with pharmaceutically acceptable organic or inorganic bases.

The present application is the National Stage under 35 U.S.C. 371 ofPCT/EP98/04924, filed Jul. 22, 1998, which designated the United Statesand was published under PCT Article 21(2) in English.

The present invention relates to use of indazol methoxyalkanoic acidsfor preparing a pharmaceutical composition active in the treatment ofdisorders characterized by production of MCP-1 protein.

European patent EP-B-0 382 276 describes a compound having the formula:

wherein

R and R′, the same as or different from each other, are H or an alkylhaving from 1 to 5 carbon atoms,

R′″ is H or an alkyl having from 1 to 4 carbon atoms,

and, when R′″ is H, salts thereof with pharmaceutically acceptableorganic or inorganic bases.

The above-mentioned patent additionally specifies that the compoundhaving the formula (I) possesses an analgesic activity.

Furthermore, European patent EP-B-0 510 748 describes the use of thecompound having the formula (I) for preparing a pharmaceuticalcomposition active in the treatment of autoimmune diseases.

It has now been found that the compound having the formula (I) is alsoactive in reducing production of MCP-1 protein.

As already known, MCP-1 protein (Monocyte Chemotactic Protein-1) is achemokine belonging to the β subfamily of the chemokines. It possesses astrong chemotactic activity for monoctyes and also acts on Tlymphocytes, mastocytes and basophils.

Other chemokines belonging to the β subfamily are, for example: MCP-2(Monocyte Chemotactic Protein-2), MCP-3, MCP4, MIP-1α and MIP-1β, RANTESand protein 1309.

The β subfamily differs in structure from the a subfamily; in fact,whilst the first two cysteines of the chemokines of the a subfamily areseparated by an interposed amino acid, the first two cysteines of the βsubfamily are adjacent to each other. MCP-1 is produced by several typesof cells (leucocytes, platelets, fibroblasts, endothelial cells).

Of all the known chemokines, MCP-1 shows the highest specificity inrespect of monocytes and macrophages, for which it is not only anattracting factor but also a stimulus of activation, thus inducing aprocess of production of superoxides and arachidonic acid, as well asbeing a stimulus of amplification of phagocytic activity.

Secretion of chemokines in general and especially of MCP-1 is typicallyinduced by numerous factors such as, for example, interleukin-1 (IL-1),interleukin-2 (IL-2), TNFα (Tumor Necrosis Factor α), γ-interferon andbacterial lipopolysaccharide (LPS).

In the human, MCP-1 has been found in a large number of diseases withacute or chronic course not classified in homogeneous categories bytraditional medicine: for example, interstitial lung disorders (ILD),vasculitis and atherosclerosis.

In interstitial lung disorders, MCP-1 released by pulmonary endothelialcells, attracts and activates competent cells with consequent release ofmediators which damage the alveolar structures of the lung.

In vasculitis, MCP-1 is released by the endothelial cells of the vasafollowing harmful stimuli and attracts and activates monocytes and othercell types which become responsible for damage to the vascular wall.

In atherosclerosis, MCP-1 is produced by the vascular endotheliumfollowing damage to the vascular smooth muscle cells. MCP-1 attractsmonocytes which initially adhere to the arterial wall and then migratethrough the walls, contributing to formation of atheroma by stimulatingproliferation of smooth muscle cells.

The therapies currently used in these disorders, because they actupstream of the pathological phenomena, are aspecific and very oftenhave numerous and at times serious side effects.

The above-mentioned therapies, moreover, only enable temporary remissionof the pathological phenomena to be obtained and their high toxicityprevents their use for prolonged periods of the kind necessary on theother hand in diseases of chronic type.

For atherosclerosis, in particular, the drugs currently used only act oncertain factors which contribute to formation of the atheroma, such ashypercholesterolaemia or hypertension, whilst having no effect on thetarget of the pathological process, i.e. the vascular wall.

Chemotactic factors in general and MCP-1 in particular are also veryimportant in cases where complications occur following surgicalinterventions such as, for example, angioplasty, atherectomy,circulatory recovery techniques, transplants, organ replacements, tissuereplacements and prosthetic implants. Onset of such complications oftenmakes it necessary for the patient to undergo further intensivetherapies or even a new intervention.

U.S. Pat. No. 5,571,713 claims a composition comprising an MCP-1antisense oligonucleotide for in vitro inhibition of production of MCP-1by mononuclear human cells and smooth muscle.

There is therefore still a strong need for a pharmaceutical compositionwhich is effective in the treatment of disorders characterized byproduction of MCP-1, e.g. atherosclerosis, vasculitis, interstitial lungdisorders due to postoperative complications of cardiovascular surgery,in transplants or organ or tissue replacements and in prostheticimplants.

The object of the present invention is therefore use of a compoundhaving the formula

where

R and R′, the same as or different from each other, are H or an alkylhaving from 1 to 5 carbon atoms,

R′″ is H or an alkyl having from 1 to 4 carbon atoms,

and, when R′″ is H, salts thereof with pharmaceutically acceptableorganic or inorganic bases,

for preparing a pharmaceutical composition for the treatment of adisorder selected from the group comprising atherosclerosis, vasculitis,interstitial lung disorders, postoperative complications incardiovascular surgery, in transplants or organ or tissue replacementsand in prosthetic implants, and characterized by an increased productionof MCP-1 protein.

Preferably R′″ is H, whilst R═R′=CH₃; this product is hereinafter called“bindarit”.

Preferably the pharmaceutical compositions according to the presentinvention are prepared in suitable dosage forms comprising an effectivedose of at least one compound having the formula (I) or a salt thereofwith a pharmaceutically acceptable base and at least onepharmaceutically acceptable inert ingredient.

Examples of suitable dosage forms are tablets, capsules, coated tablets,granules, solutions and syrups for oral administration; medicatedplaster patches for transdermal administration; suppositories for rectaladministration and sterile solutions for administration by theinjectable, aerosol or ophthalmic routes.

Further suitable dosage forms are slow release and liposome based forms,for either the oral or the injectable routes.

The dosage forms may also contain other conventional ingredients, forexample: stabilising preservatives, surfactants, buffers, salts forregulation of osmotic pressure, emulsifiers, sweeteners, coloringagents, flavourings, and the like.

If required by particular therapies, the pharmaceutical compositionaccording to the present invention may contain other pharmacologicallyactive ingredients whose concomitant administration is therapeuticallyuseful.

The amount of compound having the formula (I) or of a salt thereof witha pharmaceutically acceptable base in the pharmaceutical compositionaccording to the present invention may vary within a wide rangedepending on known factors such as, for example, the type of disease tobe treated, the severity of the disease, the patient's body weight, thedosage form, the chosen administration route, the number of dailyadministrations and the efficacy of the selected compound having theformula (I). The optimum amount can nevertheless easily and routinely bedetermined by a person skilled in the art.

Typically, the amount of compound having the formula (I) or of a saltthereof with a pharmaceutically acceptable base in the pharmaceuticalcomposition according to the present invention will be such that itensures an administration level of from 1 to 100 mg/kg/day. Preferablythe administration level is of from 5 to 50 mg/kg/day or still morepreferably of from 2 to 20 mg/kg/day.

The dosage forms of the pharmaceutical composition according to thepresent invention may be prepared according to techniques which areknown to the pharmaceutical chemist, comprising mixing, granulation,compression, dissolution, sterilization and the like.

The following examples are intended to illustrate the present inventionwithout limiting it in any way.

EXAMPLE 1 Effect of Bindarit on Production of MCP-1

The capability of bindarit to reduce production of MCP-1 by leucocytes(PBMC) stimulated by LPS was evaluated. White blood cells were isolatedby centrifugation on a Ficoll gradient and then stimulated with LPS (100ng/ml) for 24 hours in the presence or absence of scalar concentrationsof bindarit. The supernatant fluid was collected at the end and levelsof MCP-1 were measured by means of a specific immunoenzymatic test.

Table 1 shows the results obtained.

TABLE 1 Bindarit (μg/ml) 0 25 50 100 MCP-1  2.7 ± 0.51  1.5 ± 0.38*  1.6± 0.18*  0.8 ± 0.04* (ng/ml) IL-8   38 ± 4.52   30 ± 5.20   37 ± 5.36 27 ± 5.8 (ng/ml) *p < 0.01.

Table 1 shows that bindarit significantly reduces production of MCP-1induced by LPS without substantially influencing levels of IL-8produced.

EXAMPLE 2 Effect of Bindarit on Cell Attraction in the Mouse “Air Pouch”

The action of bindarit was studied in an experimental model in themouse, the said model being characterized by production of MCP-1, cellinfiltration and formation of exudate. Mice were fed ad libitum with astandard diet for rodents or with the same diet with addition ofbindarit (0.5%) for 18 days. On the twelfth day, under etheranaesthesia, sterile air (5 ml) was injected under the dorsal skin ofthe mice to form a sac. On the fifteenth day, again under etheranaesthesia, further sterile air (5 ml) was injected into the pre-formedsac. On the eighteenth day, a sterile physiological solution (1 ml) wasinjected into the sac thus obtained, or an irritant (1 ml). The saidirritant was carrageen (1%) or IL-1 (40 ng). After 5 hours for IL-1, or24 hours for carrageen, the mice were sacrificed by asphyxia with CO₂.The exudate which had developed was collected and used for the leucocytecount and for measurement of the mediators produced.

Tables 2 and 3 show the results obtained.

TABLE 2 Carrageen IL-1 Exudate (ml) Leucocytes Exudate (ml) LeucocytesControl 0.95 ± 0.30 11.2 ± 2.25 1.05 ± 0.05 11.8 ± 2.43 Bindarit 0.90 ±0.08   4.5 ± 0.64** 1.00 ± 0.01  7.5 ± 2.21* *p < 0.05; **p < 0.01.

Table 2 shows that mice treated with bindarit present a significantreduction of number of infiltrated cells (leucocytes) without presentinga reduction of volume of exudate collected.

TABLE 3 Polymorphonucleates (%) Monocytes (%) Control + IL-1 74 26Bindarit + IL-1 94 6

Table 3 shows that the effect of bindarit on cell population leads to areduction of percentage of monoctyes attracted in the sac due to theeffect produced on MCP-1.

What is claimed is:
 1. A method of treatment for atherosclerosis,comprising administering to a patient suffering from atherosclerosis aneffective MCP-1 protein reducing amount of a compound of formula

wherein R and R′ are the same or different from each other and are H oran alkyl having from 1 to 5 carbon atoms; R′″ is H or an alkyl havingfrom 1 to 4 carbon atoms; and when R′″ is H, salts thereof withpharmaceutically acceptable organic or inorganic bases.
 2. The methodaccording to claim 1, wherein R′″ is H.
 3. The method according to claim1, wherein R is methyl.
 4. The method according to claim 1, wherein R′is methyl.